The occurrence of extended-spectrum β-lactamase producing Shigella spp. in Tehran, Iran.

Background and Objectives The emergence of extended-spectrum β-lactamase (ESBL)-producing Shigella spp. is of increasing clinical concern specially in children worldwide. The aim of this study was to investigate the occurrence of extended-spectrum β-lactamase producing Shigella spp. in Tehran, Iran Materials and Methods The study included all Shigella isolates recovered from pediatric patients aged less than 12 years admitted to a major pediatric hospital in Tehran, Iran, from 2008 to 2010. Bacterial identification, antimicrobial susceptibility testing, extended spectrum β-lactamases (ESBLs) screening and confirmatory tests were performed according to the standard guidelines. Conjugal transfer experiments and plasmid analysis were also carried out. Polymerase chain reaction and sequencing were used to identify the genetic determinants responsible for ESBL production. Results Four out of 55 Shigella isolates, including three S. sonnei and one S. flexneri, showed an ESBL-positive phenotype. Plasmid transfer of the ESBL phenotype was successful for the S. flexneri isolate only. By PCR and sequencing, one S. sonnei isolate tested positive for the CMY-59 gene, while the other two S. sonnei and the S. flexneri isolates tested positive for the bla TEM-1 and bla CTX-M-15 genes. Conclusion We found the prevalence of ESBL producing Shigella isolates was higher than detection rates observed in many other countries. Our finding raise concerns about the dissemination of ESBL among the strains of endemic S. sonnei throughout the country, because this species is now the most frequently isolated Shigella species in Iran and shigellosis by such strains in the community can pose a significant threat to patients and presents a challenge for disease management.


INTRODUCTION
Infections caused by Shigella species remain a major cause of diarrheal disease associated with high morbidity and mortality in developing countries, such as Iran (1). Among the four Shigella spp., S. sonnei, is the most commonly isolated species particularly in industrialized countries (2).
Adequate antibiotic therapy is helpful in shigellosis because it may shorten the clinical course of illness, significantly reduce the risk of transmission and prevent also potentially lethal complications. However, high frequencies of resistance to commonly used antimicrobial agents have been reported worldwide in Shigella spp. Moreover, the emergence of resistance to third-generation cephalosporins (3GC) in Shigella spp. is a matter of great public health concern, mainly in developing countries (3).
There are several reports showing that the frequency of resistance against different antibiotic groups other than third-generation cephalosporins is increasing among the Shigella strains isolated in .
Here we report the identification and characterization of ESBL producing Shigella strains in Tehran, Iran.

Bacterial isolates and antimicrobial susceptibility.
The study included all Shigella isolates recovered from pediatric patients aged less than 12 years and admitted to Children's Medical Center in Tehran, Iran, from November 2008 to May 2010. A single specimen was obtained from each patient, and rectal swabs were collected from patients on the day of admission at the hospital. All strains were identified at a genus level by conventional methods according to previously described procedures (16). Serotyping of the isolates was performed by slide agglutination (Shigella Antisera, Denka Seiken, Tokyo, Japan).

Detection and characterization of ESBLs.
Screening of 3 rd generation cephalosporins (3GC) resistant Shigella isolates was performed by disk diffusion method using ceftazidime, cefotaxime, ceftriaxone and cefepime disks. According to CLSI criteria for detection of ESBL producers, each isolate with inhibition zone diameter ≤22 mm for ceftazidime or ≤27 mm for cefotaxime was considered as a potential ESBL-producer. Phenotypic confirmation of ESBL production was done by double disk synergy and combination disk tests (18).
Escherichia coli ATCC 25922 and previously characterized ESBL-positive E. coli, Klebsiellapneumoniae and AmpC-positive Enterobacter cloacae strains were used as controls for antibiotic susceptibility testing and the following ESBL analysis. Phenotypic detection of AmpC enzymes was performed by using cefoxitin supplemented with cloxacillin and E. coli ATCC 25922 according to the previously described method (19). In brief, the 30 μg cefoxitin disk was supplemented with 200 μg of cloxacillin (Sigma-Aldrich, Singapore). A strain was considered to be an AmpC producer when it showed an increase in diameter around the antibiotic disk with added cloxacillin compared to that with the cefoxitin disk alone.
A multiplex PCR assay approach was used for a rapid screening of frequently encountered β-lactamases according to the procedure previously described by Dallenneet al. (20). DNA of the isolates under study was subjected to the multiplex PCR reactions I to III in 50 µL reaction mixtures containing variable concentrations of specific-group primers and 1 U of Taq polymerase (20). Amplification was carried out as follows: initial denaturation at 94°C for 10 min; 30 cycles of 94°C for 40 s, 60°C for 40 s and 72°C for 1 min; and a final extension step at 72°C for 7 min. Amplicons were visualized on 2% agarose gels.
To identify the β-lactamase genes detected in the multiplex PCR assays, amplicons were purified using a QIA quick PCR Purification Kit (Qiagen, GmbH, Germany) and bidirectional sequencing was performed. The nucleotide sequences and the deduced protein sequences were analyzed with the BLAST and Clustal W programs (multiple sequence alignment, pairwise comparisons of sequences and dendrograms).
Resistance transfer experiments were performed for all four isolates as previously described with the rifampicin resistant Escherichia coli K12J5 as the recipient. Briefly, mixed cultures of the donors and the rifampicin resistant recipient E. coli strain were incubated at 37°C overnight. The transconjugants were selected on MacConkey agar plates supplemented with ceftazidime or cefotaxime-30 µg/ml -and rifampicin -250 µg/ml. A confirmatory test for ESBL and AmpC phenotypes and PCR detection for ESBL genes were performed on transconjugant colonies by the above mentioned procedures. Plasmids from Shigella isolates and E. coli transconjugants were extracted by an alkaline lysis extraction method (21).

RESULTS
Fifty five Shigella isolates were isolated during the study period. Of theses, 51 strains (92.7%) were susceptible to 3GC. As shown in Table 1, four strains (7.3%), including three S. sonnei and one S. flexneri showed concomitant resistance to these 3GC antibiotics and were selected for ESBL production.
Double disk synergy and combination disk tests confirmed that these four strains had ESBL-positive phenotype (Table 1). bla TEM andbla CTX-M sequences were detected in the S. flexneri and two S. sonnei isolates, whereas the third S. sonnei isolate showed a bla CMY sequence. DNA sequencing revealed that the bla TEM and bla CTX-M sequences were bla TEM-1 and bla CTX-M-
Based on the conjugation assay, only the S. flexneri isolate was able to transfer the bla gene to transconjugant, confirming that it was mediated by a plasmid of approximately 107MDa (Fig. 1). Only resistance to trimethoprim was co-transferred along with the ESBL encoding sequence.

DISCUSSION
Ampicillin and sulfamethoxazole-trimethoprim are generally indicated as the first-line drugs for therapy of shigellosis. However, in the event of disease caused by a multidrug resistant strain, second-line   (7). Unfortunately, it has been recently reported that the prevalence of resistance to some commonly used antibiotics is increasing in Shigella species in Iran (22). Class 2 integrons have proved to be at least in part responsible for the antibiotic resistance spectrum (12). Beta-lactams are the most frequently used antimicrobial agents in Iran and the ESBL producer'sorganisms have been reported frequently in several bacterial species in this country (23). Recently Tajbakhsh and her colleagues have documented the presence of bla CTX-M-15 and bla CMY-2 in six Shigella strains isolated from patients admitted to Milad Hospital, Tehran, Iran (24).
An increase in the occurrence of antimicrobial resistance, including resistance to 3GC and fluoroquinolones among Shigella spp. has been observed in several countries. To date, bla CTX-M-15 , bla CTX-M-14 , bla CTX-M-3 and bla CMY-2 genes have been reported in Shigella spp. isolates (7,25,26).
In our study, the prevalence of ESBL producing Shigella isolates, accounting for 7.3% of all Shigella isolates identified in the period under study, is higher than detection rates observed in many other countries (5,7,9). Moreover, CMY-59 has never been reported in Shigella, whereas CMY-2-type AmpC ß-lactamases has been previously detected, but infrequently. Recently, the detection of AmpC β-lactamase producer strains in Shigella spp. has been reported in Iran (24).
Among all ESBL producing strains, only the S. flexneri isolate was able to transfer the bla gene to transconjugant, indicating it was mediated by a plasmid however we did not make further attempts to obtain the transfer of resistance determinants. This could have resulted from the presence of chromosomally-mediated β-lactamase genes in the parental strains that could not be transferred to the recipients. Alternatively, they can be located on large low-copy number plasmids with a low transfer frequency.
A ceftriaxone resistant Shigella strain with a plasmid mediated CMY-2 AmpC type ESBL was also responsible for a dysentery outbreak in Taiwan (26). On the contrary, CTX-M-15 are the most common types of cefotaximases identified among Shigella isolates (3,5,6,27,28). Of special concern, plasmid mediated production of this β-lactamase is widely disseminated in Enterobacteriaceae, particularly among strains isolated from nosocomial infections, but more and more frequently from community-acquired cases.
Our findings raise concerns about the dissemination of ESBL among the strains of endemic S. sonnei throughout the country, because S. sonnei is now the most frequently isolated Shigella species in Iran and shigellosis by such strains in the community can pose a significant threat to patients and presents a challenge for disease management. Thus, active and continued surveillance of antibacterial drug resistance appears to be needed as the first step, along with a judicious use of antibiotics, to minimize the spread of ESBL producing Shigella isolates. This is especially critical for the pediatric patients for whom antibiotic therapeutic options are inherently limited.